CN106854239A - Wheat stripe rust resistance GAP-associated protein GAP TaSPX-MFS and its encoding gene and application - Google Patents
Wheat stripe rust resistance GAP-associated protein GAP TaSPX-MFS and its encoding gene and application Download PDFInfo
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Abstract
The invention discloses wheat stripe rust resistance GAP-associated protein GAP TaSPX-MFS and its encoding gene and application.The present invention be cloned into SPX genes in 54 from small the laying down of Common Wheat Varieties, is named as TaSPX-MFS, and constructs the virus induction carrier for TaSPX-MFS genes in silence wheat, reduces the expression of TaSPX-MFS genes, obtains silence TaSPX-MFS wheats.Proved by testing：After silence TaSPX-MFS wheats inoculation stripe rust CYR31, occur many strip rust bacteria sorus on wheat leaf blade, wheat plant manifests obvious stripe rust resistance and loses phenotype.Illustrate to suppress expression of the TaSPX-MFS genes in wheat in plant by means such as knockout or silences, resistance of the wheat to stripe rust can be reduced, for research stripe rust of wheat, there is provided responsive type strain, is that the research of wheat resistance genes is made contributions.
Description
Technical field
The present invention relates to wheat stripe rust resistance GAP-associated protein GAP TaSPX-MFS and its encoding gene and application, belong to raw
Thing technical field.
Background technology
In very long evolutionary process, plant generates the mechanism of complexity to recognize infecting and trigger itself and having for pathogen
The immune response of effect.Plant congenital immunity system includes Liang great branches, and one is PAMP (pathogen-associated
Molecular pattern) triggering immune system (PAMP-triggered immunity, PTI), it is by cross-film
Receptor protein PRRs (pattern recognition receptors) and PAMP early stage for mutually replying and producing to cause of disease
The Resistant reaction of bacterium；Another is the immune system (effector-triggered immunity, ETI) of effector triggering,
It is the R albumen encoded by resistant gene in plant (R genes) to being encoded by pathogen nontoxic gene (Avr genes)
Effector carry out direct or indirect identification and produce immune response (Chisholm ST, Coaker G, Day B,
Staskawicz BJ.(2006).Host-microbe interactions:shaping the evolution of the plant
immune response.Cell 124:803-814；Nimchuk Z,Eulgem T,Holt III BF,Dangl JL.(2003).
Recognition and response in the plant immune system.Annu.Rev.Genet.37:579-609)。
Hordeivirus (Barley Stripe Mosaic Virus, BSMV) is that totally 3 chains are constituted by α, β, γ
3 strand rna virus.The gene silencing system mediated using BSMV-based is employed successfully in barley and wheat function base
(Hein I, Barciszewska-Pacak M, Hrubikova K, Williamson S, Dinesen M, Soenderby in the research of cause
IE,Sundar S,Jarmolowski A,Shirasu K,Lacomme C.(2005).Virus-Induced Gene Silencing-Based
Functional Characterization of Genes Associated with Powdery Mildew Resistance in Barley.Plant
Physiology 138:2155-2164；Zhou HB,Li SF,Deng ZY,Wang XP,Chen T,Zhang JS,Chen SY,
Ling HQ,Zhang AM,Wang DW,Zhang XQ.(2007).Molecular analysis of three new receptor-like
kinase genes from hexaploid wheat and evidence for their participation in the wheat hypersensitive
response to stripe rust fungus infection.The Plant Journal 52,420–434.；Wang G,Wei X,Fan R,
Zhou H,Wang X.Yu C,Dong L,Wang X,Kang Z,Ling H,Shen Q,Wang D,Zhang X.
(2011)Molecular analysis of common wheat genes encoding three types of cytosolic heat
shock protein 90(hsp90):functional involvment of ctosolic hsp90s in the control of wheat
seedling growth and disease resistance.New Phytol.,191:418-431)。
When BSMV infects plant, plant is carried out by the corresponding siRNA of DCL4 and DCL2 generations to viral RNA
Montage, thus resists the injury of virus.But when carrying exogenous genetic fragment in viral RNA, its siRNA can also lead
The silence of plant endogenous genes is caused, phenotype (the Dekeris A., Gallego-Bartolome of plant gene silencing is thus produced
J.,Bao J.,Kasschau K.,Carrington J.,Voinnet O.(2006).Hierarchical Action and Inhibition
of Plant Dicer-Like Proteins in Antiviral Defense.Science.313(5783),68-71).Molecular Virology
Research shows, does not influence it to infect activity to plant when exogenous sequences are inserted after γ b genes.
The content of the invention
The technical problems to be solved by the invention are how to regulate and control plant stripe rust resistance.
In order to solve the above technical problems, present invention firstly provides with plant stripe rust resistance GAP-associated protein GAP, institute of the present invention
There is provided the entitled TaSPX-MFS with plant stripe rust resistance GAP-associated protein GAP, be following a)-e) protein：
A) amino acid sequence is the protein shown in sequence 2 in sequence table；
B) amino acid sequence is the protein shown in sequence 4 in sequence table；
C) amino acid sequence is the protein shown in sequence 6 in sequence table；
D) shown in the sequence 2 or the N-terminal and/or C-terminal connection label of sequence 4 or the protein shown in sequence 6 is obtained
Fused protein；
E) by sequence 2 Suo Shi or sequence 4 or amino acid sequence shown in sequence 6 are by one or several amino acid residues
Substitution and/or missing and/or the protein with identical function that obtains of addition.
It is a further object to provide the biomaterial with above-mentioned albumen qualitative correlation.
What the present invention was provided is following A 1 with the biomaterial of above-mentioned albumen qualitative correlation) to A12) in any one：
A1 the nucleic acid molecules of above-mentioned protein) are encoded；
A2 A1) is contained) expression cassette of the nucleic acid molecules；
A3 A1) is contained) recombinant vector of the nucleic acid molecules；
A4 A2) is contained) recombinant vector of the expression cassette；
A5 A1) is contained) recombinant microorganism of the nucleic acid molecules；
A6 A2) is contained) recombinant microorganism of the expression cassette；
A7 A3) is contained) recombinant microorganism of the recombinant vector；
A8 A4) is contained) recombinant microorganism of the recombinant vector；
A9 A1) is contained) the transgenic plant cells system of the nucleic acid molecules；
A10 A2) is contained) the transgenic plant cells system of the expression cassette；
A11 A3) is contained) the transgenic plant cells system of the recombinant vector；
A12 A4) is contained) the transgenic plant cells system of the recombinant vector.
In above-mentioned relevant biological material, A1) nucleic acid molecules are following 1) -5) shown in gene：
1) its coded sequence is cDNA molecules or the DNA molecular shown in sequence 1；
2) its coded sequence is cDNA molecules or the DNA molecular shown in sequence 3；
3) its coded sequence is cDNA molecules or the DNA molecular shown in sequence 5；
1) or 2) or 3) 4) have in 75% or more than 75% homogeneity, and coding with the nucleotide sequence for limiting
State the cDNA molecules or genomic DNA molecule of protein；
5) under strict conditions with 1) or 2) or 3) or 4) nucleotide sequence hybridization for limiting, and encode above-mentioned egg
The cDNA molecules or genomic DNA molecule of white matter.
New application it is a still further object of the present invention to provide above-mentioned protein or with above-mentioned protein relevant biological material.
The invention provides above-mentioned protein or with above-mentioned protein relevant biological material in plant stripe rust resistance is regulated and controled
Application.
Stripe rust resistance reduction is being cultivated present invention also offers above-mentioned protein or with above-mentioned protein relevant biological material
Genetically modified plants in application.
In above-mentioned application, the plant is monocotyledon, and the monocotyledon is specially wheat.
It is a still further object of the present invention to provide a kind of method of the genetically modified plants for cultivating stripe rust resistance reduction.
The method of the genetically modified plants of the cultivation stripe rust resistance reduction that the present invention is provided includes that above-mentioned protein table will be suppressed
The material that reaches is imported in recipient plant, the step of obtain genetically modified plants；The stripe rust resistance of the genetically modified plants is low
In the recipient plant.
In the above method, the material for suppressing above-mentioned protein expression is following A) or B)：
A) BSMV viral vectors α, BSMV viral vectors β and shown from 5 ' 255-442, ends containing sequence 3
DNA molecular BSMV viral vectors γ；
B) BSMV viral vectors α, BSMV viral vectors β and contain sequence 3 from 5 ' end 2067-2250 institutes
The BSMV viral vectors γ of the DNA molecular for showing.
In the above method, the stripe rust resistance of the genetically modified plants is embodied in as follows less than the recipient plant
A1) or a2)：
A1) the strip rust bacteria silk of genetically modified plants is more long than recipient plant；
A2) the strip rust bacteria sorus of genetically modified plants is more than recipient plant.
In the above method, the recipient plant is monocotyledon；The monocotyledon is specially wheat.
Final object of the present invention is to provide the material for suppressing above-mentioned protein expression.
The material of the above-mentioned protein expression of suppression that the present invention is provided is following A) or B)：
A) BSMV viral vectors α, BSMV viral vectors β and shown from 5 ' 255-442, ends containing sequence 3
DNA molecular BSMV viral vectors γ；
B) BSMV viral vectors α, BSMV viral vectors β and contain sequence 3 from 5 ' end 2067-2250 institutes
The BSMV viral vectors γ of the DNA molecular for showing.
The present invention be cloned into SPX genes in 54 from small the laying down of common wheat (Triticum aestivum L.) kind, by it
TaSPX-MFS is named as, and constructs the virus induction carrier for TaSPX-MFS genes in silence wheat,
The expression of TaSPX-MFS genes is reduced, silence TaSPX-MFS wheats are obtained.Proved by testing：Silence
After TaSPX-MFS wheats inoculation stripe rust CYR31, occur many strip rust bacteria sorus on wheat leaf blade, wheat is planted
Strain manifests obvious stripe rust resistance and loses phenotype.Illustrate by knocking out or the means such as silence are come in suppressing wheat
Expression of the TaSPX-MFS genes in plant, can reduce resistance of the wheat to stripe rust, otherwise speculate, in wheat
In overexpression TaSPX-MFS genes can improve resistance of the wheat for stripe rust, for research stripe rust of wheat,
Responsive type strain is provided, is that the research of wheat resistance genes is made contributions.
Brief description of the drawings
Fig. 1 is BSMV-based VIGS systemic vectors structural representations, as external source base by taking TaPDS genes as an example
Reversely inserted because of fragment at the terminator of γ chain γ b genes mutation.
Fig. 2 is the expression of TaSPX-MFS after gene silencing.
Fig. 3 is small 54 pairs of resistances changes of strip rust bacteria microspecies CYR31 of laying down after virus induction TaSPX-MFS gene silencings
Change.
It is thin that Fig. 4 is that virus induction restructuring hordeivirus infects the growth of internal strip rust bacteria silk and host after plant
Born of the same parents' reaction observation.
Specific embodiment
Experimental technique used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
It 54 is that in August, 2000 passes through Shaanxi Province's Crop breed audit that Common Wheat Varieties in following embodiments are small to lay down
Committee's Approved variety, in document " Wu Huijie, Wei Xuening, Zhou Xinli, Cao Shuanhe, Li Hongwei, Wang Xianping, Tong Yi
It is flat, the well study of the Chinese classic, Zhang Xiangqi.Small 54 and small 81 High temperature resistances and its pass with chitinase gene expression of laying down of laying down
System.Wheat crops journal, 2007,27 (6):Mistake disclosed in 1117-1122 ", the public can from the Chinese Academy of Sciences heredity with
Developmental Biology research is obtained.
High-fidelity Pyrobest Taq, Premix Ex Taq in following embodiments, RNase inhibitor, various limitations
Property restriction endonuclease, reverse transcription reagent box, pMD18-T Vector be purchased from precious biology (TaKaRa) company in Dalian；DNA
Purifying QIAquick Gel Extraction Kit is purchased from Tiangeng biotech company；Transcription related kit is biological purchased from Pu Luomaige (Beijing)
Technology Co., Ltd..
BSMV viral vectors (including BSMV viral vectors α, BSMV viral vectors β in following embodiments
With BSMV viral vectors γ) it is to be presented by Andy Jackson professors (University of California, Berkeley)
Give, in document " Petty I, Hunter B, Wei N, Jackson A. (1989) .Infectious barley stripe
mosaicvirus RNA transcribed in vitro from full-length genomic cDNA clones.Virology,
171,342-349 " mistake disclosed in, the public can be obtained from Chinese Academy of Sciences's heredity with Developmental Biology research.
Strip rust bacteria microspecies CYR31 in following embodiments document " Li XH, Fan RC, Fu SL, Wei B, Feng J,
Zheng Q,Wang XP,Han FP,Zhang XQ.(2015).Development of Triticum
aestivum-Leymus mollis translocation lines and identification of resistance to stripe rust.
Mistake disclosed in Journal of Genetics and Genomic 42 (3), 129-132. ", the public can plant from the Chinese Academy of Agricultural Sciences
Thing Protective strategy is bought.
GFP (BSMV in following embodiments:GFP) plasmid is in document " Holzberg S., Brosio P., Gross
C., Pogue G. (2002) Barley stripe mosaic virus-induced gene silencing in a monocot plant.
Plant J.,30:Mistake disclosed in 315-327 ", the public can be obtained from Chinese Academy of Sciences's heredity with Developmental Biology research.
GKP Buffer in following embodiments contain 50mM glycine (glycine), 30mM K2HPO41% (matter
Amount percentage) bentonite (bentonite), 1% (mass percent) diatomite (celite).
The acquisition of embodiment 1, TaSPX-MFS genes
1st, the acquisition of cDNA
Small 54 seedling leaves of laying down of Common Wheat Varieties of growth 7 days are taken, is put into after cleaning through DEPC treatment, sterilizing
In mortar afterwards, grind, add TRIzol (Invitrogen) grindings, homogenate, room temperature is placed 5 minutes, plus
Enter chloroform 2 times, take supernatant, use isopropanol precipitating total serum IgE, after being air-dried, be dissolved in appropriate DEPC water.
Reverse transcription is carried out with the primer (TaKaRa) of band polyT and obtains cDNA.
2nd, PCR amplifications
It is template with the cDNA that step 1 is obtained, using forward primer TaSPX-MFS-F and reverse primer
TaSPX-MFS-R enters performing PCR amplification, obtains pcr amplification product.Primer sequence is as follows：
TaSPX-MFS-F：5′-CGAAGTATTGTGCTTGAGTTGATTG-3′
TaSPX-MFS-R：5′-CCTGCTAGCATTTGCTAGCTTGATACCAC-3′；
Above-mentioned pcr amplification reaction condition：94 DEG C of 1min, then 94 DEG C of 15s, 58 DEG C of 15s, 72 DEG C of 2min 30
Circulation, last 72 DEG C of 5min.
3rd, electrophoresis and sequencing
The pcr amplification product that above-mentioned steps 2 are obtained is entered into row agarose gel electrophoresis, electrophoresis result shows, PCR
Amplification obtains the fragment of size about 2.3kb, and pcr amplification product is connected into pMD18-T carriers (TaKaRa
Biotech it is used to be sequenced on), sequence assembly is carried out to sequencing result using DNAman softwares, as a result shows：It is above-mentioned
Lay down in 54 that clone obtains 3 different cDNA sequences altogether from Common Wheat Varieties are small, 3 gene order similitudes are non-
Chang Gao, nucleotide homology 96.6~98.3%, all between 97.3-99% incite somebody to action by amino acid sequence similarity
3 genes are referred to as TaSPX-MFS genes, are respectively designated as TaSPX-MFS-6A genes, TaSPX-MFS-6B
Gene and TaSPX-MFS-6D genes, wherein, sequence in the nucleotide sequence of TaSPX-MFS-6A genes such as sequence table
It is OFR from 5 ' end 1-2082 shown in row 1, the amino acid sequence of its coding is as shown in sequence 4 in sequence table；
The nucleotide sequence of TaSPX-MFS-6B genes is OFR from 5 ' end 1-2082 as shown in sequence 2 in sequence table,
The amino acid sequence of its coding is as shown in sequence 5 in sequence table；The nucleotide sequence of TaSPX-MFS-6D genes such as sequence
It is OFR, sequence 6 in the amino acid sequence of its coding such as sequence table from 5 ' end 1-2082 in list shown in sequence 3
It is shown.
The analysis of embodiment 2, the acquisition of silence TaSPX-MFS wheats and its stripe rust resistance
First, the acquisition of silence TaSPX-MFS wheats
1st, the acquisition of gene silencing fragment
(1) using TaSPX-MFS-6D genes in step 1 as template, using SPXdm-VF1：
5’-TACGCTAGCCATACTCACGGAACATTCCG-3’；SPXdm-VR1：5’-CCTGCTAGC
CACGAGTCGAGACGTAATAA-3 ' enters performing PCR amplification, and amplification obtains the fragment of 188bp (from sequence 3
5 ' end 255-442 nucleotides), be named as SPXdm-VR1；
(2) using TaSPX-MFS-6D genetic fragments in step 1 as template, using 3UTR-VF1：5’-
TACGCTAGCTTATAACACCCTGTACTGAC-3’；3UTR-VR1:5’-
CCTGCTAGCATTTGCTAGCTTGATACCAC-3 ', amplification obtains the fragment of 184bp (from sequence 3
5 ' 2067-2250 the nucleotides in end) it is named as 3UTR-VR1.
2nd, the structure of BSMV recombinant viral vectors
According to conventional molecular biological means, referring to " fine works molecular biology experiment guide, 2001, Science Press,
Yan Ziying, Wang Hailin is translated " in method carry out vector construction.Comprise the following steps that：
Between the NheI restriction enzyme sites of the SPXdm-VR1 insertion BSMV viral vectors γ that above-mentioned steps 1 are obtained,
And keep the other sequences of BSMV viral vectors γ constant, obtain recombinant vector VIGS-SPXdm.
Between the NheI restriction enzyme sites of the 3UTR-VR1 insertion BSMV viral vectors γ that above-mentioned steps 1 are obtained, and
Keep the other sequences of BSMV viral vectors γ constant, obtain recombinant vector VIGS-3UTR (Fig. 1, with TaPDS
As a example by illustrate exogenous genetic fragment insertion position and direction).
BSMV viral vectors α, β and BSMV:GFP together constitutes virus system BSMV:GFP viral vectors.
BSMV viral vectors α, β and recombinant vector VIGS-SPXdm are together constituted can silence TaSPX-MFS
The virus system BSMV of gene:SPXdm viral vectors.
BSMV viral vectors α, β and recombinant vector VIGS-3UTR are together constituted can silence TaSPX-MFS bases
The virus system BSMV of cause:3UTR viral vectors.
3rd, BSMV in-vitro transcriptions
(1) with MluI digestion BSMV viral vectors α, BSMV:GFP, VIGS-SPXdm, VIGS-3UTR,
With SpeI digestion BSMV viral vectors β, digestion products are respectively obtained.
(2) in-vitro transcription is carried out by template of the digestion products in (1), respectively obtains the BSMV diseases of in-vitro transcription
Poisonous carrier α, the BSMV viral vectors β of in-vitro transcription, the BSMV of in-vitro transcription:GFP, in-vitro transcription
The VIGS-3UTR of VIGS-SPXdm and in-vitro transcription.In-vitro transcription is reacted according to AmpliCap-MaxTM T7
High Yield Message Maker Kit (Epicentre) specification is operated：RNase-free water X μ L,
1.5 μ g, 10 × Transcription Buffer of linearization plasmid, 2 μ L, Cap/NTP PreMix 8 μ L, 100mM DTT
2 μ L, Amplicap-MaxTMThe μ L of T7Enzyme Solution 2, reaction cumulative volume 20 μ L, 42 DEG C of reaction 2.5h,
Transcription product is put -70 DEG C and is saved backup.
4th, BSMV inoculations
Whne it is small lay down 54 grow to two leaf stage when, by 8 μ l BSMV virus mixed liquor (sterilizing ultra-pure water dilute 3 times after body
The BSMV viral vectors α of outer transcription, the BSMV viral vectors β of in-vitro transcription, the BSMV of in-vitro transcription:GFP
Mixed in equal amounts, adds isometric 2 × GKP Buffer) smear and be inoculated on the second open and flat leaf of plant, after 10 minutes
Blade face is sprayed with sterilizing ultra-pure water, preservative film moisturizing is covered 24 hours, normal condition culture is switched to afterwards, turned
BSMV:GFP strains.
Whne it is small lay down 54 grow to two leaf stage when, by 8 μ l BSMV virus mixed liquor (sterilizing ultra-pure water dilute 3 times after body
The BSMV viral vectors α of outer transcription, the BSMV viral vectors β of in-vitro transcription, the VIGS-SPXdm of in-vitro transcription
Mixed in equal amounts, adds isometric 2 × GKP Buffer) smear and be inoculated on the second open and flat leaf of plant, after 10 minutes
Blade face is sprayed with sterilizing ultra-pure water, preservative film moisturizing is covered 24 hours, normal condition culture is switched to afterwards, turned
BSMV:SPXdm strains.
Whne it is small lay down 54 grow to two leaf stage when, by 8 μ l BSMV virus mixed liquor (sterilizing ultra-pure water dilute 3 times after body
The BSMV viral vectors α of outer transcription, the BSMV viral vectors β of in-vitro transcription, the VIGS-3UTR of in-vitro transcription
Mixed in equal amounts, adds isometric 2 × GKP Buffer) smear and be inoculated on the second open and flat leaf of plant, after 10 minutes
Blade face is sprayed with sterilizing ultra-pure water, preservative film moisturizing is covered 24 hours, normal condition culture is switched to afterwards, turned
BSMV:3UTR strains.
It is control to be inoculated with 1 × GKP (buffer simulations inoculation), obtains MOCK (control).
Turn BSMV:SPXdm and turn BSMV:3UTR strains are the silence of stripe rust resistance reduction of the present invention
The lines of TaSPX-MFS.
5th, the identification of silence TaSPX-MFS wheats
Detected by RT-PCR and turn BSMV:SPXdm and turn BSMV:TaSPX-MFS genes in 3UTR strains
The situation of silence.Specific method is as described below：
(1) MOCK (control) that is obtained after virus inoculation in extraction step 4 respectively, BSMV is turned:GFP strains,
Turn BSMV:SPXdm and turn BSMV:The total serum IgE of 3UTR strains, reverse transcription obtains cDNA.
(2) cDNA with above-mentioned steps (1) acquisition carries out RT-PCR as template using following primer, respectively
The expression quantity of detection wheat TaSPX-MFS-6A, TaSPX-MFS-6B and TaSPX-MFS-6D.It is with Actin
Reference gene, primer sequence is as follows：
SPX-MFS-6A：qTaSPX-MFS-6A-F：5′-CTTCCGGTCAACGTCATCGTT-3′；
qTaSPX-MFS-6A-R：5′-CGACACGACGTACTGAGCTT-3′；
SPX-MFS-6B：qTaSPX-MFS-6B-F：5′-CTCCATCGCCGCGACGTTT-3′；
qTaSPX-MFS-6B-R：5′-GCTAGCTTGATACCACAAGCC-3′；
SPX-MFS-6D：qTaSPX-MFS-6D-F：5′-TCCGGTCAACGTCATCGTG-3′；
qTaSPX-MFS-6D-R：5′-ACAGGTTGACCCCTTCAAGA-3′；
Actin：qActin-F:5′-CGAGCTCCGTGTCGCA 3′；
qActin-R:5′-GAGGAAGCGTGTATCCCTCATAG-3′。
Result is as shown in Figure 2：MOCK (control) and turn BSMV:Three TaSPX-MFS genes in GFP strains
The expression of (TaSPX-MFS-6A genes, TaSPX-MFS-6B genes and TaSPX-MFS-6D genes) is not bright
Aobvious change, and turn BSMV:SPXdm and turn BSMV:Three TaSPX-MFS genes in 3UTR strains
The expression of (TaSPX-MFS-6A genes, TaSPX-MFS-6B genes and TaSPX-MFS-6D genes) is obtained
It is obvious to suppress, reach the effect of gene silencing.
2nd, the stripe rust resistance analysis of silence TaSPX-MFS wheats
1st, the inoculation of strip rust bacteria microspecies CYR31
MOCK (control), turn BSMV:GFP strains, turn BSMV:SPXdm strains, turn BSMV:3UTR
Strain inoculation BSMV virus mixed liquors, culture starts inoculation strip rust bacteria microspecies CYR31 after 20 days.Specific steps are such as
Under：Wheat seedling to be seeded is put into inoculation bucket, will with the Toween20 solution of 0.1% (volumn concentration)
Uniformly squirted around wheat seedling and inoculation bucket, then carry out sweeping for CYR31 microspecies and smear inoculation, afterwards again with 0.1%
Toween20 is uniformly squirted, and bung is obturaged moisturizing with plastic paper.After inoculation, it is dark that seedling carries out (10 DEG C) of low temperature
Treatment 24h, is infected with the success for ensureing strip rust bacteria.Phjytotron growth is then moved within second day, day temperature is
15-18 DEG C, 11-14 DEG C of evening, humidity is 80% or so, and intensity of illumination is 6000Lux, and light application time is 16h/d.
Phenotype is observed after 15 days.
Result such as Fig. 3 institutes：Inoculation and response type grade scale press method (Li ZQ, the Shang HS. of Li and Shang descriptions
(1989).Wheat rusts and their control.Shanghai Science and Technology Press,Shanghai,
China.), 0-2 grades is disease-resistant, and 3-4 grades is susceptible.Turn BSMV:SPXdm strains and turn BSMV:3UTR strains are inoculated with
Occur many strip rust bacteria sorus (3 grades) after strip rust bacteria CYR31, on wheat leaf blade, and MOCK (control) and turn
BSMV:Many necrotic plaques (0 grade) are then occurred in that in GFP strains, hypersensitivity (HR) is generated, MOCK is (right
According to) and turn BSMV:The resistance of the stripe rust of GFP strains is higher than to turn BSMV:SPXdm strains and turn BSMV:3UTR
Strain.The above results illustrate that TaSPX-MFS take part in resistance processes of the wheat to stripe rust opportunistic pathogen CYR31.
2nd, the morphologic observation of mycelia
In order to more fully understand that TaSPX-MFS participates in the plant pathology histology machine of small 54 high temperature induction strip rust resistemces of laying down
System, using fluorescence microscope to inoculation CYR314 days after MOCK (control), turn BSMV:GFP strains, turn
BSMV:SPXdm strains, turn BSMV:The growth spread scenarios and host's leaf of strip rust bacteria in 3UTR strain blade materials
Meat cell effect is observed, and Hyphal length is counted.Research material order connects the bacterium blade of 4 days, leaf
Piece treatment is with reference to " Wang CF, Huang LL, Buchenauer H, Han QM, Zhang HC, Kang ZS. (2007)
Histochemical stidies on the accumulation of reactive oxygen species(O2-and H2O2)in the
incompatible and compatible interaction of wheat-Puccinia striiformis f.sp.tritici.Physiol.&
Mol.Plant Pathol.,71:Method in 230-239 ".Comprise the following steps that：
The leaf section of 2 centimeter lengths of clip is put into fixed, decolouring in 95% ethanol solution, and observation is forwarded to saturation hydration
In chloral solution overnight.Then, preserved in 50% glycerite.Observation is with statistical method with reference to " Wang XJ, Tang
CL,Zhang HC,Xu JR,Liu B,Lv J,Han DJ,Huang LL,Kang ZS.(2011)TaDAD2,a
negative regulator of programmed cell death,is important for the interaction between wheat
and the stripe rust fungus.Mol.Plant Microbe Interact.,24:Method in 79-90 ".Leaf after decolouring
Section Olympus BX-51 Differential interference contrast microscopes (differential interference contrast microscope,
DIC) length to strip rust bacteria infection court and invasion mycelia is observed, and is recorded.Using fluorescence microscope necrosis
The autofluorescence of mesophyll cell, and record necrosis point area.
Result is as shown in Figure 4：Turning BSMV:SPXdm strains, turn BSMV:The mesophyll cell of 3UTR strains is bad
It is dead little, the extension without limitation strip rust bacteria silk, strip rust bacteria generates secondary hyphae, and (Fig. 4-c, d), Hyphal length shows
Work is longer than MOCK (control), turns BSMV:The length (Fig. 4-e) of mycelia in GFP strains.MOCK (control),
Turn BSMV:In GFP strain blades, strip rust bacteria silk is shorter, in the site contacted with primary hyphae, host's mesophyll cell
Produce necrosis, limit mycelia extension (Fig. 4-a, b).
Claims (10)
1. protein, is following a)-e) protein：
A) amino acid sequence is the protein shown in sequence 2 in sequence table；
B) amino acid sequence is the protein shown in sequence 4 in sequence table；
C) amino acid sequence is the protein shown in sequence 6 in sequence table；
D) shown in the sequence 2 or the N-terminal and/or C-terminal connection label of sequence 4 or the protein shown in sequence 6 is obtained
Fused protein；
E) by sequence 2 Suo Shi or sequence 4 or amino acid sequence shown in sequence 6 are by one or several amino acid residues
Substitution and/or missing and/or the protein with identical function that obtains of addition.
2., with the biomaterial of the albumen qualitative correlation described in claim 1, be following A 1) to A12) in any
Kind：
A1) the nucleic acid molecules of the protein described in coding claim 1；
A2 A1) is contained) expression cassette of the nucleic acid molecules；
A3 A1) is contained) recombinant vector of the nucleic acid molecules；
A4 A2) is contained) recombinant vector of the expression cassette；
A5 A1) is contained) recombinant microorganism of the nucleic acid molecules；
A6 A2) is contained) recombinant microorganism of the expression cassette；
A7 A3) is contained) recombinant microorganism of the recombinant vector；
A8 A4) is contained) recombinant microorganism of the recombinant vector；
A9 A1) is contained) the transgenic plant cells system of the nucleic acid molecules；
A10 A2) is contained) the transgenic plant cells system of the expression cassette；
A11 A3) is contained) the transgenic plant cells system of the recombinant vector；
A12 A4) is contained) the transgenic plant cells system of the recombinant vector.
3. relevant biological material according to claim 2, it is characterised in that：A1) nucleic acid molecules be as
Lower 1) -5) gene shown in：
1) its coded sequence is cDNA molecules or the DNA molecular shown in sequence 1；
2) its coded sequence is cDNA molecules or the DNA molecular shown in sequence 3；
3) its coded sequence is cDNA molecules or the DNA molecular shown in sequence 5；
1) or 2) or 3) 4) there is 75% or more than 75% homogeneity, and coding power with the nucleotide sequence for limiting
Profit requires the cDNA molecules or genomic DNA molecule of the protein described in 1；
5) under strict conditions with 1) or 2) or 3) or 4) nucleotide sequence hybridization for limiting, and coding right will
Seek the cDNA molecules or genomic DNA molecule of the protein described in 1.
4. the relevant biological material described in the protein or Claims 2 or 3 described in claim 1 is in regulation and control plant bar
Application in rust resistance.
5. the relevant biological material described in the protein or Claims 2 or 3 described in claim 1 is cultivating stripe rust
Application in the genetically modified plants of resistance reduction.
6. the application according to claim 4 or 5, it is characterised in that：The plant is monocotyledon.
7. it is a kind of cultivate stripe rust resistance reduction genetically modified plants method, including will suppress claim 1 described in
The material of protein expression is imported in recipient plant, the step of obtain genetically modified plants；The bar rust of the genetically modified plants
Sick resistance is less than the recipient plant.
8. method according to claim 7, it is characterised in that：The protein suppressed described in claim 1
The material of expression is following A) or B)：
A) BSMV viral vectors α, BSMV viral vectors β and containing sequence 3 from shown in 255-442,5' ends
DNA molecular BSMV viral vectors γ；
B) BSMV viral vectors α, BSMV viral vectors β and sequence 3 from 2067-2250,5' ends institute is contained
The BSMV viral vectors γ of the DNA molecular for showing.
9. the method according to claim 7 or 8, it is characterised in that：The stripe rust resistance of the genetically modified plants
Following a1 is embodied in less than the recipient plant) or a2)：
A1) the strip rust bacteria silk of genetically modified plants is more long than recipient plant；
A2) the strip rust bacteria sorus of genetically modified plants is more than recipient plant；
The recipient plant is monocotyledon.
10. suppress the material of the protein expression described in claim 1, be following A) or B)：
A) BSMV viral vectors α, BSMV viral vectors β and containing sequence 3 from shown in 255-442,5' ends
DNA molecular BSMV viral vectors γ；
B) BSMV viral vectors α, BSMV viral vectors β and sequence 3 from 2067-2250,5' ends institute is contained
The BSMV viral vectors γ of the DNA molecular for showing.
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| CN108004251A (en) * | 2018-02-02 | 2018-05-08 | 山东农业大学 | The breeding method of application and Rust resistance bacterium wheat of the wheat stripe rust PSTG_11438 genes in stripe rust prevention |
| CN108559753A (en) * | 2018-02-02 | 2018-09-21 | 山东农业大学 | The breeding method of application and Rust resistance bacterium wheat of the wheat stripe rust PSTG_17694 genes in stripe rust prevention |
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| CN108795927A (en) * | 2018-07-02 | 2018-11-13 | 河南农业大学 | The clone of common wheat gene TaSPX3 coded sequences and its application |
| CN114621967A (en) * | 2022-05-16 | 2022-06-14 | 西北农林科技大学深圳研究院 | Wheat receptor protein kinase gene TaLEMK1.1 and application thereof |
| CN116286724A (en) * | 2023-05-15 | 2023-06-23 | 西北农林科技大学深圳研究院 | Lectin receptor protein TaLecRLK2 and encoding gene and application thereof |
| CN116286724B (en) * | 2023-05-15 | 2023-08-25 | 西北农林科技大学深圳研究院 | Lectin receptor protein TaLecRLK2 and encoding gene and application thereof |
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